The present invention relates to the isolation and use of nucleic acid molecules for control of gene expression in plants, specifically novel plant promoters.
One of the goals of plant genetic engineering is to produce plants with agronomically important characteristics or traits. Recent advances in genetic engineering have provided the requisite tools to transform plants to contain and express foreign genes (Kahl et al. (1995) World Journal of Microbiology and Biotechnology 11:449-460). Particularly desirable traits or qualities of interest for plant genetic engineering would include but are not limited to resistance to insects and other pests and disease-causing agents, tolerances to herbicides, enhanced stability, yield, or shelf-life, environmental tolerances, and nutritional enhancements. The technological advances in plant transformation and regeneration have enabled researchers to take pieces of DNA, such as a gene or genes from a heterologous source, or a native source, but modified to have different or improved qualities, and incorporate the exogenous DNA into the plant""s genome. The gene or gene(s) can then be expressed in the plant cell to exhibit the added characteristic(s) or trait(s). In one approach, expression of a novel gene that is not normally expressed in a particular plant or plant tissue may confer a desired phenotypic effect. In another approach, transcription of a gene or part of a gene in an antisense orientation may produce a desirable effect by preventing or inhibiting expression of an endogenous gene.
Isolated plant promoters are useful for modifying plants through genetic engineering to have desired phenotypic characteristics. In order to produce such a transgenic plant, a vector that includes a heterologous gene sequence that confers the desired phenotype when expressed in the plant is introduced into the plant cell. The vector also includes a plant promoter that is operably linked to the heterologous gene sequence, often a promoter not normally associated with the heterologous gene. The vector is then introduced into a plant cell to produce a transformed plant cell, and the transformed plant cell is regenerated into a transgenic plant. The promoter controls expression of the introduced DNA sequence to which the promoter is operably linked and thus affects the desired characteristic conferred by the DNA sequence.
Since the promoter is a 5xe2x80x2 regulatory element that plays an integral part in the overall expression of a gene or gene(s), it would be advantageous to have a variety of promoters to tailor gene expression such that a gene or gene(s) is transcribed efficiently at the right time during plant growth and development, in the optimal location in the plant, and in the amount necessary to produce the desired effect. In one case, for example, constitutive expression of a gene product may be beneficial in one location of the plant, but less beneficial in another part of the plant. In other cases, it may be beneficial to have a gene product produced at a certain developmental stage of the plant, or in response to certain environmental or chemical stimuli. The commercial development of genetically improved germplasm has also advanced to the stage of introducing multiple traits into crop plants, often referred to as a gene stacking approach. In this approach, multiple genes conferring different characteristics of interest can be introduced into a plant. It is important when introducing multiple genes into a plant, that each gene is modulated or controlled for optimal expression and that the regulatory elements are diverse, to reduce the potential of gene silencing which can be caused by recombination of homologous sequences. In light of these and other considerations, it is apparent that optimal control of gene expression and regulatory element diversity are important in plant biotechnology.
The proper regulatory sequences must be present and in the proper location with respect to the DNA sequence of interest, for the newly inserted DNA to be transcribed and thereby, if desired translated into a protein in the plant cell. These regulatory sequences include but are not limited to a promoter, a 5xe2x80x2 untranslated leader, and a 3xe2x80x2 polyadenylation sequence. The ability to select the tissues in which to transcribe such foreign DNA, and the time during plant growth in which to obtain transcription of such foreign DNA is also possible through the choice of appropriate promoter sequences that control transcription of these genes.
A variety of different types or classes of promoters can be used for plant genetic engineering. Promoters can be classified on the basis of range or tissue specificity. For example, promoters referred to as constitutive promoters are capable of transcribing operatively linked DNA sequences efficiently and expressing said DNA sequences in multiple tissues. Tissue-enhanced or tissue-specific promoters can be found upstream and operatively linked to DNA sequences normally transcribed in higher levels in certain plant tissues or specifically in certain plant tissues. Other classes of promoters would include but are not limited to inducible promoters which can be triggered by external stimuli such as chemical agents, developmental stimuli, or environmental stimuli. Thus, the different types of promoters desired can be obtained by isolating the upstream 5xe2x80x2 regulatory regions of DNA sequences which are transcribed and expressed in a constitutive, tissue-enhanced, or inducible manner.
The technological advances of high-throughput sequencing and bioinformatics has provided additional molecular tools for promoter discovery. Particular target plant cells, tissues, or organs at a specific stage of development, or under particular chemical, environmental, or physiological conditions can be used as source material to isolate the mRNA and construct cDNA libraries. The cDNA libraries are quickly sequenced and the expressed sequences catalogued electronically. Using sequence analysis software, thousands of sequences can be analyzed in a short period, and sequences from selected cDNA libraries can be compared. The combination of laboratory and computer-based subtraction methods allows researchers to scan and compare cDNA libraries and identify sequences with a desired expression profile. For example, sequences expressed preferentially in one tissue can be identified by comparing a cDNA library from one tissue to cDNA libraries of other tissues and electronically xe2x80x9csubtractingxe2x80x9d common sequences to find sequences only expressed in the target tissue of interest. The tissue enhanced sequence can then be used as a probe or primer to clone the corresponding full-length cDNA. A genomic library of the target plant can then be used to isolate the corresponding gene and the associated regulatory elements, including promoter sequences.
Multiple promoter sequences which confer a desired expression profile such as promoters capable of regulating expression of operably linked genes in multiple tissues can be isolated by selectively comparing cDNA libraries of target tissues of interest with non-target or background cDNA libraries to find the 5xe2x80x2 regulatory regions associated with the expressed sequences in those target libraries. The isolated promoter sequences can be used for selectively modulating expression of any operatively linked gene and provide additional regulatory element diversity in a plant expression vector in gene stacking approaches.
The present invention provides nucleic acid sequences, which comprise regulatory sequences located upstream of the 5xe2x80x2 end of plant DNA structural coding sequences that are transcribed in multiple tissues of interest and shown in SEQ ID NOS: 53-75.
In one aspect, the present invention provides nucleic acid sequences comprising a sequence selected from the group consisting of SEQ ID NOS: 53-75 or any fragments, regions, or cis elements of the sequence that are capable of regulating transcription of operably linked DNA sequences.
The present invention also provides nucleic acid sequences comprising a sequence selected from the group consisting of SEQ ID NOS: 53-75 that are promoters.
Another aspect of the present invention relates to the use of at least one fragment, region, or cis element thereof of the disclosed 5xe2x80x2 promoter sequences that can be combined to create novel promoters or used in a novel combination with another heterologous regulatory sequence to create a hybrid or chimeric promoter capable of modulating transcription of an operably linked DNA sequence.
Hence, the present invention relates to the use of nucleic acid sequences disclosed in SEQ ID NOS: 53-75, or any fragment, region, or cis elements of the disclosed sequences that are capable of regulating transcription of a DNA sequence when operably linked to the DNA sequence. Therefore, the invention not only encompasses the sequences as disclosed in SEQ ID NOS: 53-75, but also includes any truncated or deletion derivatives, or fragments or regions thereof which are capable of functioning independently as a promoter including cis elements that are capable of functioning as regulatory sequences in conjunction with one or more regulatory sequences when operably linked to a transcribable sequence.
The present invention thus encompasses a novel promoter or a hybrid or chimeric promoter comprising a nucleic acid of SEQ ID NOS: 53-75. The hybrid or chimeric promoters can consist of any length fragments, regions, or cis elements of the disclosed sequences of SEQ ID NOS: 53-75 combined with any other transcriptionally active minimal or full-length promoter. For example, a promoter sequence selected from SEQ ID NOS: 53-75 may be combined with a CaMV 35S or other promoter to construct a novel hybrid promoter. A minimal promoter can also be used in combination with the nucleic acid sequences of the present invention. A novel promoter also comprises any promoter constructed by engineering the nucleic acid sequences disclosed in SEQ ID NOS: 53-75 or any fragment, region, or cis element of the disclosed sequences in any manner sufficient to transcribe an operably linked DNA sequence.
Another aspect of the present invention relates to the ability of the promoter sequences of SEQ ID NOS: 53-75, or fragments, regions, or cis elements thereof to regulate transcription of operably linked transcribable sequences in multiple tissues. Fragments, regions, or cis elements of SEQ ID NOS: 53-75 that are capable of regulating operably linked DNA sequences in certain tissues may be isolated from the disclosed nucleic acid sequences of SEQ ID NOS: 53-75 and used to engineer novel promoters that confer a desired expression profile.
The present invention also encompasses DNA constructs comprising the disclosed sequences as shown in SEQ ID NOS: 53-75 or any fragments, regions, or cis elements thereof, including novel promoters generated using the disclosed sequences or any fragment, region, or cis element of the disclosed sequences.
The present invention also includes any cells and transgenic plants containing the DNA disclosed in the sequences as shown in SEQ ID NOS: 53-75, or any fragments, regions, or cis elements thereof.
The present invention also provides a method of regulating transcription of a DNA sequence comprising operably linking the DNA sequence to any promoter comprising a nucleic acid comprising all or any fragment, region or cis element of a sequence selected from the group consisting of SEQ ID NOS: 53-75.
In a another embodiment the present invention provides a method regulating expression of DNA sequences in multiple tissues by operably linking a sequence selected from the group consisting of SEQ ID NOS: 53-75, or any fragment, region, or cis element of the disclosed sequences to any transcribable DNA sequence. The fragments, regions, or cis elements of the disclosed promoters as shown in SEQ ID NOS: 53-75 can be engineered and used independently in novel combinations including multimers, or truncated derivatives and the novel promoters can be operably linked with a transcribable DNA sequence. Alternatively the disclosed fragments, regions, or cis elements of the disclosed sequences can be used in combination with a heterologous promoter including a minimal promoter to create a novel hybrid or chimeric promoter and the novel hybrid promoter can be operably linked to a transcribable DNA sequence.
The present invention also provides a method of making a transgenic plant by introducing into the cell of a plant a DNA construct comprising: (i) a promoter comprising a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NOS: 53-75, or fragments, regions, or cis elements thereof, and operably linked to the promoter, (ii) a transcribable DNA sequence and (iii) a 3xe2x80x2 untranslated region.
The present invention also provides a method of isolating at least one 5xe2x80x2 regulatory sequence of a desired expression profile from a target plant of interest by evaluating a collection of nucleic acid sequences of expressed sequence tags (ESTs) derived from one or more cDNA libraries prepared from a plant cell type of interest, comparing EST sequences from at least one target plant cDNA library and one or more non-target cDNA libraries of ESTs from a different plant cell type, subtracting common EST sequences found in both target and non-target libraries, designing gene-specific primers from the remaining ESTs after the subtractions which are representative of the targeted expressed sequences, and isolating the corresponding 5xe2x80x2 flanking and regulatory sequences, which includes promoter sequences from a genomic library prepared from the target plant using the gene specific primers.